Both types 1 & 2 diabetes (T1DM, T2DM) result from the inability of -cells to secrete sufficient insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate -cell mass. Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from growth factors (GFs) & nutrients to regulate cell growth & proliferation. Recent studies have determined that inhibition of glycogen synthase kinase-3 (GSK-3) & tuberous sclerosis complex 2 (TSC2) are required for mTOR activation. Our studies have shown that GSK-3 inhibition stimulates mTOR-mediated DNA synthesis & cell cycle progression in rodent & human islets. The highly reproducible stimulation of mTOR-mediated DNA synthesis by lithium, a GSK-3 inhibitor, in human islets is remarkable since nutrients are rarely effective. Since GSK-3 inhibition is also integral to Wnt/2-catenin transcriptional regulation, we propose to evaluate possible interactions of this pathway with mTOR signaling. Our goal is to enhance the growth & proliferative capacity of adult -cells by appropriately regulating both mTOR & the canonical Wnt signaling pathways using GSK-3 inhibitors, Wnt agonists & nutrients. Specific aim 1 is to establish the functions of the GSK-3/TSC/mTOR & the canonical Wnt/GSK-3/2-catenin pathways in primary adult rodent & human islets by A) identifying the molecular & cellular targets & the ability of GSK-3 inhibitors, Wnt agonists, nutrients & rapamycin to modulate these pathways, B) elucidating the role of these pathways in the growth, proliferation & function of -cells & C) determining if a differential capacity to affect feedback inhibition of growth factor (GF) signaling distinguishes nutrients from GSK-3 inhibitors. Using isolated islets, we will utilize siRNA of GSK-3, -catenin & mTOR, study Wnt- dependent TCF/LEF1 transcription using transgenic TOPGAL mice & evaluate GF signaling and GSK-3 activity after chronic nutrient or GSK-3 inhibitor treatment. Specific aim 2 is to determine if the mitochondrial transition pore (MTP) & nitric oxide (NO) are required for GSK-3 & mTOR signaling by A) examining if RNA interference of nitric oxide synthase (NOS) isoforms or MTP components, VDAC & ANT, alter the physiological responses of rodent & human islets to nutrients or GSK-3 inhibition, B) evaluating the ability of GSK-3 inhibitors, Wnt agonists or nutrients to regulate NOS expression & NO production & C) investigating how exogenous NO affects the MTP & -cell growth, proliferation & function. Specific aim 3 is to determine whether manipulation of GSK-3 and mTOR signaling will promote -cell function, growth & proliferation in a T1DM islet transplantation model by pre-culture of human or rodent islets with GSK-3 inhibitors or Wnt agonists 1 rapamycin prior to transplanting a marginal dose of islets into STZ-diabetic SCID mice. Understanding the co-regulation of the GSK-3/TSC/mTOR & Wnt/GSK-3/-catenin pathways will provide new strategies to enhance growth & proliferation of adult -cells, serve important unmet medical needs of T1DM & T2DM & provide basic mechanistic knowledge of how nutrients affect these pathways.